Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa of humans. Although most H. pylori-infected persons remain asymptomatic, potentially serious sequelae of infection include gastric adenocarcinoma, duodenal ulceration, gastric ulceration, and gastric lymphoma. Gastric cancer is the second leading cause of cancer-related death worldwide, and H. pylori has been classified as a type I carcinogen by the World Health Organization. One of the major secreted proteins of H. pylori is a toxin known as VacA. VacA causes multiple alterations in gastric epithelial cells, and inhibits activation and proliferation of T lymphocytes. Most cellular effects of VacA are dependent on its ability to form anion-selective membrane channels. There is a high level of genetic variation among vacA alleles from unrelated H. pylori strains, and the encoded VacA proteins exhibit marked differences in their ability to cause alterations in human cells. The molecular basis for the observed differences in activities is not yet completely understood. A large body of literature indicates that H. pylori strains containing certain forms of vacA (termed s1, i1, or m1) are associated with a higher risk of gastric cancer or peptic ulcer disease than are strains containing other forms of vacA (termed s2, i2, or m2). Thus, VacA is considered to be an important H. pylori virulence factor. The long-term goals of this work are to understand the mechanisms by which H. pylori infection can lead to disease, to understand the basis for variation in clinical outcomes among H. pylori-infected persons, and to develop effective means for prevention and treatment of illnesses associated with H. pylori infection. The specific aims are (i) to investigat VacA structural features that are required for intracellular toxin activity and membrane channel formation, (ii) to analyze differences in functional properties of VacA proteins encoded by different H. pylori strains, and (iii) to identify and analyze host cell components that are requird for VacA cytotoxicity. Methods will include cryo-electron microscopy, crystallography, molecular genetics, and analysis of gene trap and shRNA libraries. This work is relevant not only for the study of H. pylori-associated diseases, but will also increase our understanding of bacterial pore-forming toxins, chloride-conducting membrane channels, beta- helical passenger domains secreted by an autotransporter pathway, and protein targeting of mitochondria. PUBLIC HEALTH RELEVANCE: A bacterium known as Helicobacter pylori colonizes the stomach in about half of all humans. Most H. pylori-infected persons do not develop any symptoms related to this infection, but some develop gastric cancer or peptic ulcer disease. The long-term goals of this research are to understand the mechanisms by which H. pylori infection can lead to disease, to understand the basis for variation in clinical outcomes among H. pylori-infected persons, and to develop effective means for prevention and treatment of illnesses associated with H. pylori infection.